Conference Agenda

Fumihiro Ito, Kelsey Mainard, Jingjing Yan, Robin Erickson, Andrew Nguyen, Aabha Vora, Lijuan Feng and Wanhe Li

The molecular clock drives the circadian rhythm and its behavioral and physiological outputs through tightly regulated gene expression. Histone posttranslational modifications (PTMs) alter chromatin structure and recruitments of transcriptional factors, thereby epigenetically regulating gene expression. Many types of PTMs, including acetylation and methylation, exhibit rhythms at promoters, enhancers, and gene bodies, hence modifying the accessibility of genes to transcriptional machinery. Histone monoaminylation is a recently recognized kind of PTM, where neurotransmitters, such as excessive dopamine or serotonin resulting from pathological conditions, are covalently bonded to the histone tail. This process regulates neuronal transcription. Most recently, a previously unreported histone monoaminylation called histaminylation was identified. Characterized as a PTM, histaminylation cycles in mice’s hypothalamic tuberomammillary nucleus (TMN) throughout their sleep/wake cycle. The functions of these emerging types of PTMs in epigenetic regulation in the nervous system, both in healthy and diseased brain states, are being uncovered, making them highly intriguing. In this study, we built a neurogenetics toolbox to study histone modifications using the model organism Drosophila melanogaster. We found that perturbing histone monoaminylation resulted in a unique sleep phenotype. This phenotype reflects a defect in sleep maintenance during the nighttime but not sleep initiation, because sleep deprivation can induce normal sleep rebound. Subsequently, we employed a comprehensive set of molecular, genetics, and genomics approaches to further explore histone monoaminylation-dependent sleep regulation. We identified the neural circuit in which the dynamics of gene expression are regulated through epigenetic mechanisms. Since the monoamine biochemistry and histone proteins are remarkably conserved between humans and flies, the discovery of histone monoaminylation-dependent sleep regulation may reveal a conserved sleep regulatory mechanism in an epigenetic setting.

Department of Biology, Texas A&M University

Illia Pimenov¹, Courtney M. MacMullen², Justine David¹, Ronald L. Davis², Anna Phan¹

Learning and memory research has focused its attention mainly on genes that promote learning. Meanwhile, limiting memory formation has been largely overlooked until more recently. Stromalin has been identified as a learning suppressor that functioned by restricting the synaptic vesicle (SV) pool size in dopamine neurons (DANs) in Drosophila melanogaster. While we have previously established SV numbers to be critical for learning and memory, the mechanism of SV numbers regulation remains unexplored. To advance our cognizance of this conundrum, a DAN-specific RNA-Seq with knocked down stromalin was performed to identify the genes that act downstream of it and mediate its effects on SV pool size. We obtained RNAi lines to significantly differentially expressed genes and performed a primary aversive olfactory memory screen and a secondary SV marker (Syt:eGFP) screen in DANs. Initial results suggested 5 promising candidates: CG17698, nep1, su(z)12, cox7c, and ttm2. Based on the validation experiments, we outlined two primary candidates: nep1 and su(z)12. Upon examining mRNA levels after su(z)12 knockdown using Nanostring’s nCounter, we have postulated that nep1 levels are not regulated by a transcriptional regulator su(z)12. Hence, our current hypothesis is that stromalin regulates the transcription of nep1 to suppress learning via constricting SV numbers. We are testing it by looking into whether overexpression of nep1 can rescue stromalin knockdown effects on memory. Besides, we plan on looking at the dopamine release from DANs with silenced stromalin and overexpressed nep1 using in vivo functional imaging with the dopamine sensor GRAB_DA to further test our hypothesis.

¹Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada ²Department of Neuroscience, Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, FL, USA

Funding Support: Natural Sciences and Engineering Research Council (NSERC) grant RGPIN-2020-04009, Mitacs Globalink Research Internship Award, Mitacs Globalink Research Award, Mitacs Globalink Graduate Fellowship Award.

RC Simamora1,2, JA Krawitz1, L Herzig1, RA Jain1

Sensorimotor gating is a process where the nervous system filters incoming stimuli to reduce responsiveness to irrelevant stimuli. Major forms of sensorimotor gating include prepulse inhibition (PPI) and habituation. Through PPI, response to a strong “pulse” stimulus is attenuated when it is immediately preceded by a weaker “prepulse” stimulus. During habituation, responses to repeated innocuous stimuli are diminished or abolished. Both processes are disrupted in conditions with complex genetic underpinning such as autism spectrum disorder (ASD), though the degree to which these forms of gating are controlled by shared or divergent genetic factors is not fully understood. The ap2s1 gene, which encodes a subunit of the endocytosis Adaptor Protein Complex 2 (AP-2), has recently been linked to ASD, though its behavioral contributions to this condition have been unclear.

Here we use zebrafish mutations in multiple AP-2 complex subunits to test the requirements for this complex in sensorimotor gating. We find that ap2s1, as well as ap2a1 which encodes another AP-2 subunit, are both required for acoustically-evoked habituation. We optimize an assay for robust PPI in zebrafish larvae, and validate that similar to mammalian systems, acute disruption of NMDA receptors with MK-801 robustly disrupts both habituation and PPI in zebrafish. Similar to habituation, we find that ap2s1 and ap2a1 are also both required for PPI, indicating an essential role of AP-2 in multiple forms of sensorimotor gating. By investigating the genetic underpinnings of sensorimotor gating, we establish zebrafish as a model for examining how ap2s1 might influence conditions such as ASD.

1Department of Biology, Haverford College, Haverford, PA, USA. 2Department of Human Genetics, Emory University, Atlanta, GA, USA.

Funding Support: NIH R15EY031539

Barbara D. Fontana¹, Neha Rajput¹, Jacob Hudock¹, Dea Kanani¹, and Justin W. Kenney¹

Fear is a fundamental emotional state that is highly conserved across the animal kingdom. Despite the intrinsic importance of fear for survival, its behavioral manifestation varies between individuals of a species where the choice of the best response can be the difference between life and death. However, we know little about the biological basis for individual differences in fear behavior. To plumb the depths of behavioral variation, we used adult zebrafish as a model organism. Fish were trained to associate a new environment with fear by exposing them to conspecific alarm substance (CAS), an ethologically relevant chemical stimulus released from the epithelial cells of injured fish to alert nearby animals to danger. Animals were tracked using DeepLabCut, and we trained a random forest machine learning model to identify different behaviors (e.g., freezing, bursting, and erratic movements) to greater than 95% accuracy. After collecting fear learning and memory data from over 400 animals from four different in bred strains (AB, TU, TL, and WIK) and both sexes, we used unsupervised machine learning to identify four distinct behavioral clusters: (1) low fear responsivity, (2) increased erratic behavior, (3) high freezing interspersed with increased erratic behavior, and (4) high freezing interspersed with normal swimming behavior. We found that strain, but not sex, had a strong influence on the type of fear behavior exhibited. Finally, we identified the neural basis for these individual differences by mapping whole-brain c-fos expression using in situ HCR, tissue clearing, light-sheet microscopy, image registration, and network analysis.

¹Department of Biological Sciences, Wayne State University, Detroit, MI, 48202

Funding support: NIGMS R35GM142566

Alyson Blount¹, Emmanuelle Palmieri¹, & Laurie Sutton¹

GPR158 is a G protein coupled receptor (GPCR) that is highly expressed in the brain, is upregulated by stress exposure, and is implicated in cognition. Standard cognitive tests can induce stress as they require multiple tests, experimenter handling, and are limited as they are only a snapshot of behavior. To overcome these caveats, automated home cage monitoring systems allow for longitudinal, repetitive, objective, and consistent measurements of mouse behavior in the absence of experimenter handling while in a social and familiar environment. Here we used the Intellicage, an automated operant home cage, and standard Barnes Maze test to elucidate the cognitive effects of GPR158 on age and sex. In the Intellicage, we assessed levels of spatial and preference learning in GPR158 global knockouts (KO) and wildtype littermates. Aged mice (≥ 14 months), regardless of sex or genotype, showed deficiencies in spatial learning and preference learning compared to young adult mice (3-6 months) in both the Intellicage and Barnes Maze. In young mice, both genotypes and sexes showed similar levels of spatial and preference learning in the Intellicage, in contrast to our Barnes Maze results and previous literature indicating that GPR158 KOs have cognitive deficits. Overall, automatic home cage monitoring allows for further unbiased long term behavioral phenotyping that may uncover subtle behavioral differences in sex, age, and genotype. Our findings suggest that traditional stressful cognitive testing paradigms may affect behavioral phenotyping and further research is needed to characterize mouse models in more social and naturalistic conditions.

Department of Biological Sciences, University of Maryland Baltimore County (UMBC)

V Liu¹, TS Andrews^2,3, AR Khan^1,4,5, JC Lau^1,4,6

The zona incerta (ZI) is a promising neuromodulatory target for alleviating Parkinsons’ Disease (PD) motor symptoms. Recent research reveals a dopaminergic ZI subregion, named the A13, is implicated in emotion processing deficits and chronic pain, whereas its stimulation effectively restores locomotion deficits in mouse PD models. Despite its significance, the A13 has not been visualized in humans, preventing translation to clinical applications. To address this challenge, we aim to identify the A13 by integrating transcriptomics and MRI-derived phenotypes in humans. We hypothesize that the A13 is enriched in dopaminergic neurons, and correlates with elevated quantitative susceptibility mapping (QSM; iron deposition measure) values in PD patients. We acquired whole brain spatially registered microarray data from the Allen Human Brain Atlas (n=6), and applied dimensionality reduction techniques to identify ZI clusters and explore their biological relevance. In parallel, we recruited a cohort of healthy (n=42, age 20-70) and PD patients (n = 46, age 51 - 73) for 7T MRI to generate averaged T1-map and QSM images in MNI space. We identified 5 distinct clusters along the rostral-caudal axis in the human ZI, including a putative A13 located in the rostromedial ZI. Consistent with the definition of the A13 region in mice, this region is enriched with dopaminergic and GABAergic signatures, along with genes implicated in PD pathogenesis. In addition, we found that the rostral ZI is enriched with GABAergic signatures, associated with neurogenesis regulation. By integrating macroscale imaging with transcriptomics, we not only highlight the unique organization within the ZI, but also present potential pathways for improved neuromodulatory targeting and enhancing patient outcomes.

¹ Imaging Research Laboratories, Department of Neuroscience, Robarts Research Institute, Western University, London, Canada
².Department of Biochemistry, Western University, London, Canada
^3.Department of Computer Science, Western University, London, Canada
⁴.School of Biomedical Engineering, Western University, London, Canada
⁵.Department of Medical Biophysics, Schulich School of Medicine and Dentistry, Western University, London, Canada
^6.Department of Clinical Neurological Sciences, Division of Neurosurgery, Western University, London, Canada

Funding Support: Department of Clinical Neurological Sciences, Western University